EMS26123

For The Histological Diagnosis Of Fungi In Tissue Sections

Components:

  • Fungi in tissue, includes:

  • Chromic Acid, 10%

  • Sodium Bisulfite, 1%

  • Silver Nitrate, 5% Aqueous

  • Methenamine, 3% Aqueous

  • Sodium Borate, 5% Aqueous

  • Gold Chloride, 0.2%

  • Sodium Thiosulfate, 0.2% Aqueous

  • Harris’ Hematoxylin

  • Acetic Acid 3%

  • Ammonia Water, 0.25%

  • Eosin Y, 1% Alcoholic

Fixation:

10% Buffered Neutral Formalin (NBF)

Sections

3-4µm

Staining Procedure:

  1. Deparaffinise and hydrate to distilled water.

  2. Oxidise in Chromic Acid, 10% for 10 minutes.

  3. Rinse with 3-4 changes of tap water.

  4. Rinse in Sodium Bisulfite, 1% for 1 minute to remove any residual Chromic Acid.

  5. Wash in running water, 5-10 minutes.

  6. Rinse with 3-4 changes of distilled water.

  7. Place in freshly mixed working methenamine Silver Nitrate Solution in a water bath @ 58-60 °C, for 15-25 minutes or until sections turn yellowish brown.

Methenamine Silver Nitrate Stock:
Silver Nitrate, 5% Aq - 5ml
Methenamine, 3% Aq - 100ml
Use chemically clean glassware. Shake well to dissolve precipitate. Store in refrigerator.

Methenamine Silver Nitrate Working solution:
Methenamine Silver Nitrate Stock Solution - 25ml
Water - 25ml
Sodium Borate, 5% Aq - 4ml

  1. Rinse in 6 changes of distilled water.

  2. Tone in Gold Chloride, 0.2% for 2-5 minutes.

  3. Rinse in 3-4 changes of distilled water.

  4. Remove unreduced Silver with Sodium Thiosulfate, 2% for 2-5 minutes.

  5. Wash thoroughly in tap water for 10 minutes.

  6. Place in Harris' Haematoxylin for 10 minutes.

  7. Rinse in 3-4 changes of tap water.

  8. Place in Acetic Acid, 3%) for 1 minute.

  9. Rinse in 2 changes tap water.

  10. Place in Ammonia Water, 0.25% 10 minutes.

  11. Rinse in 2 changes tap water.

  12. Place in 95% alcohol, 1 minute.

  13. Place in Eosin Y, 1% Alcoholic for 5 minutes.

  14. Dehydrate in 95% alcohol , absolute alcohol and clear in xylene, 2 changes each.

  15. Mount with Permount.

Stain Results:

Organisms

Blue-Black

Background

Rose

References:

Lamps, Laura W. et al A Combined Hematoxylin and Eosin/ M ethenamine Silver Stain for the Histological Diagnosis of Fungi in Tissue Sections. , J. of Histotechnology 23:4 p. 341-345